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Inflammation mediated angiogenesis in chronic lymphocytic leukemia

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Abstract

Chronic inflammation has been identified in leukemias as an essential regulator of angiogenesis. B-chronic lymphocytic leukemia (CLL) cells secrete high levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1 alpha (HIF1α). The aim was to assess the role of inflammation in activation of angiogenic factors: endothelial nitric oxide synthase (eNOS), HIF1α and VEGF via proliferation related signaling pathways and VEGF autocrine control. We isolated mononuclear cells (MNC) and CD19+ cells from peripheral blood of 60 patients with CLL. MNC were treated with pro-inflammatory interleukin-6 (IL-6) and VEGF, in combination with inhibitors of JAK1/2 (Ruxolitinib), mTOR (Rapamycin), NF-κB (JSH23), SMAD (LDN-193189) and PI3K/AKT (Ly294002) signaling pathways, to evaluate eNOS, VEGF and HIF1α expression by immunoblotting, immunocytochemistry and RT-qPCR. Also, we investigated IL-6 dependent neovascularization in human microvascular endothelial cells (HMEC-1) in co-culture with MNC of CLL. The angiogenic factors eNOS, VEGF and HIF1α had significantly higher frequencies in MNC of CLL in comparison to healthy controls (p < 0.001) and CD19+ cells of CLL. IL-6 increased the quantity of HIF1α (p < 0.05) and VEGF positive cells in the presence of JSH23 (p < 0.01). VEGF increased HIF1α (p < 0.05), and decreased eNOS gene expression (p < 0.01) in MNC of CLL. VEGF significantly (p < 0.001) increased the number of HIF1α positive MNC of CLL, prevented by inhibitors of JAK1/2, PI3K and mTOR signaling pathways. VEGF stimulation of SMAD (p < 0.05) and STAT5 (p < 0.01) signaling has been prevented by inhibitors of JAK1/2, mTOR, PI3K and SMAD signaling, individually (p < 0.01) or mutually (p < 0.001). Also, we showed that MNC of CLL and IL-6 individually stimulate neovascularization in co-culture with HMEC-1, without a cumulative effect. We demonstrated elevated angiogenic factors in CLL, while VEGF and IL-6 independently stimulated HIF1α. VEGF stimulation of HIF1α was mostly mTOR dependent, while IL-6 stimulation was NF-κB dependent.

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Data available on request by email to corresponding author.

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Funding

This research was supported by The Ministry of Science, Technological Development and Innovation of the Republic of Serbia grant number 451–03-66/2024–03/200015.

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Author notes

  1. Olivera Mitrović-Ajtić and Emilija Živković contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Oncology, Institute for Medical Research, National Institute of Republic of Serbia, University of Belgrade, Dr. Subotića 4, 11129, Belgrade, Serbia

    Olivera Mitrović-Ajtić, Emilija Živković, Tijana Subotički, Miloš Diklić, Dragoslava Đikić, Milica Vukotić, Teodora Dragojević & Vladan P. Čokić

  2. Lymphoma Center, Clinic for Hematology, University Clinical Center of Serbia, Belgrade, Serbia

    Vojin Vuković & Darko Antić

  3. Faculty of Medicine, University of Belgrade, Belgrade, Serbia

    Darko Antić

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  1. Olivera Mitrović-Ajtić
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Contributions

Conceptualization, V.Č., D.A. and O.M.A.; methodology, T.S., O.M.A., M.D. and D.Đ.; formal analysis, T.S., O.M.A., E.Ž. and M.D.; investigation, T.S., O.M.A., M.V., T.D. and D.Đ.; data curation, O.M.A. and M.V., writing—original draft preparation, O.M.A., T.S., V.V., and V.Č.; writing—review & editing, O.M.A. and V.Č.; visualization, O.M.A. and E.Ž.; supervision, V.Č.; project administration, V.Č. All authors have read and agreed to the published version of the manuscript.

Corresponding author

Correspondence to Olivera Mitrović-Ajtić.

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The study was approved by the Ethics Committee of the University Clinical Centre of Serbia, Belgrade (decision number 187/4) and the Ethics Committee of the Institute for Medical Research, Belgrade (decision number EO 117/2016).

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Mitrović-Ajtić, O., Živković, E., Subotički, T. et al. Inflammation mediated angiogenesis in chronic lymphocytic leukemia. Ann Hematol (2024). https://doi.org/10.1007/s00277-024-05781-1

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